Structural determination of the polysaccharide isolated from biofilms produced by a clinical strain of Klebsiella pneumoniae

Klebsiella pneumoniae are Gram negative opportunistic pathogens producing capsular (K) polysaccharides. Seventy-seven different K antigens have been described and they are the basis for K serotyping. Capsular polysaccharides are important virulence factors and have a relevant role for the structure of biofilm communities. Nevertheless, little information is available on the polysaccharides produced in biofilm matrices by Klebsiella spp. In the present study, a clinical isolate of Klebsiella pneumoniae was grown both on cellulose membranes deposited on agar plates, where it formed an adherent biofilm, and in liquid medium, where it formed floating biofilms (flocs). Extraction and purification of the polysaccharide fraction showed that only one main carbohydrate polymer was present in both adherent biofilms and flocs. Composition and linkage analysis, Smith degradation followed by ESI-MS, 1D and 2D NMR spectroscopy revealed that the polysaccharide belong to the type K24 and has the following structure: [2)[beta-D-Manp(1-4)]-alfa-D-GlcpA-(1-3)-alfa-D-Manp-(1-2)-alfa-D-Manp-(1-3)-beta-D-Glcp-(1 ]

The effect of mushroom culture filtrates on the inhibition of mycotoxins produced by Aspergillus flavus and Aspergillus carbonarius

Two of the mycotoxins of greatest agroeconomic significance are aflatoxin B-1 (AFB(1)), and ochratoxin A (OTA). It has been reported that extracts from some wood-decaying mushrooms, such as Lentinula edodes and Trametes versicolor showed the ability to inhibit AFB(1) or OTA biosynthesis. Therefore, in our study, a wide screening of 42 isolates of different ligninolytic mushrooms was assayed for their ability to inhibit the synthesis of OTA in Aspergillus carbonarius and AFB(1) in Aspergillus flavus, in order to find a metabolite that can simultaneously inhibit both mycotoxins. The results showed that four isolates produce metabolites able to inhibit the synthesis of OTA, and 11 isolates produced metabolites that inhibited AFB(1) by >50%. Two strains, the Trametes versicolor strain TV117 and the Schizophyllum commune strain S.C. Ailanto, produced metabolites able to significantly inhibit (>90%) the synthesis of both mycotoxins. Preliminary results suggest that the mechanism of efficacy of the S. commune rough and semipurified polysaccharides could be analogous to that found previously for Tramesan(R), by enhancing the antioxidant response in the target fungal cells. The overall results indicate that S. commune's polysaccharide(s) could be a potential agent(s) in biological control and/or a useful component of the integrated strategies able to control mycotoxin synthesis

Structural analysis of the O-acetylated O-polysaccharide isolated from Salmonella Paratyphi A and used for vaccine preparation

Salmonella paratyphi A is increasingly recognized as a common cause of enteric fever cases and there are no licensed vaccines against this infection. Antibodies directed against the O-polysaccharide of the lipopolysaccharide of Salmonella are protective and conjugation of the O-polysaccharide to a carrier protein represents a promising strategy for vaccine development. O-Acetylation of S. paratyphi A O-polysaccharide is considered important for the immunogenicity of S. paratyphi A conjugate vaccines. Here, as part of a programme to produce a bivalent conjugate vaccine against both S. typhi and S. paratyphi A diseases, we have fully elucidated the O-polysaccharide structure of S. paratyphi A by use of HPLC\u2013SEC, HPAEC\u2013PAD/CD, GLC, GLC\u2013MS, 1D and 2D-NMR spectroscopy. In particular, chemical and NMR studies identified the presence of O-acetyl groups on C-2 and C-3 of rhamnose in the lipopolysaccharide repeating unit, at variance with previous reports of O-acetylation at a single position. Moreover HR-MAS NMR analysis performed directly on bacterial pellets from several strains of S. paratyphi A also showed O-acetylation on C-2 and C-3 of rhamnose, thus this pattern is common and not an artefact from O-polysaccharide purification. Conjugation of the O-polysaccharide to the carrier protein had little impact on O-acetylation and therefore should not adversely affect the immunogenicity of the vaccine

Structure of the capsular polysaccharide of the KPC-2-producing Klebsiella pneumoniae strain KK207-2 and assignment of the glycosyltransferases functions

Klebsiella pneumoniae strain KK207-2 was isolated in 2010 froma bloodstreaminfection of an inpatient at an Italian hospital. It was previously found to produce the KPC-2 carbapenemase and to belong to clade 1 of sequence type 258. Genotyping of the conserved wzi and wzc genes from strain KK207-2 yielded contrasting results: the wzc-based method assigned the cps207\u20132 to a new K-type, while the wzi-based method assigned it to the known K41 K-type. In order to resolve this contradiction, the capsular polysaccharide of K. pneumoniae KK207-2 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives, ESI-MS of both partial hydrolysis and Smith degradation derived oligosaccharides, andNMR spectroscopy of oligosaccharides, and the lithium degraded, native and de-O-acetylated polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KK207-2 capsular polysaccharide: OAc 6 [3)-\u3b2-D-Gal-(1-4)-\u3b2-D-Glc-(1-]n 4 I 1 \u3b2-D-Glcp-(1-6)-\u3b1-D-Glcp-(1-4)-\u3b2-D-GlcpA-(1-6)-\u3b1-D-Glcp The polysaccharide contains about 0.60 acetyl groups per repeating unit on C6 of the Gal residue. The reactions catalysed by each glycosyltransferase in the cpsKK207-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens

Multifrequency STD NMR unveils the interactions of antibiotics with Burkholderia multivorans biofilm exopolysaccharide

Biofilms confine bacterial cells within self-produced matrices, offering advantages such as protection from antibiotics and entrapment of nutrients. Polysaccharides are major components in these macromolecular assemblies, and their interactions with other chemicals are of high relevance for the benefits provided by the biofilm 3D molecular matrix. NMR is a powerful technique for the study and characterization of the interactions between molecules of biological relevance. In this study, we have applied multifrequency saturation transfer difference (STD) NMR and DOSY NMR approaches to elucidate the interactions between the exopolysaccharide produced by Burkholderia multivorans C1576 (EpolC1576) and the antibiotics kanamycin and ceftadizime. The NMR strategies presented here allowed for an extensive characterization at an atomic level of the mechanisms behind the implication of the EpolC1576 in the recalcitrance phenomena, which is the ability of bacteria in biofilms to survive in the presence of antibiotics. Our results suggest an active role for EpolC1576 in the recalcitrance mechanisms toward kanamycin and ceftadizime, though through two different mechanisms

Rapid generation of Shigella flexneri GMMA displaying natural or new and cross-reactive O-Antigens

Generalized modules for membrane antigens (GMMA) are exosomes released from engineered Gram-negative bacteria and represent an attractive vaccine platform for the delivery of the O-Antigen (OAg), recognized as the key target for protective immunity against several pathogens such as Shigella. Shigella is a major cause of disease in Low- and Middle-Income countries and the development of a vaccine needs to deal with its large serotypic diversity. All S. flexneri serotypes, except serotype 6, share a conserved OAg backbone, corresponding to serotype Y. Here, a GMMA-producing S. flexneri scaffold strain displaying the OAg backbone was engineered with different OAg-modifying enzymes, either individually or in combinations. This strategy rapidly yielded GMMA displaying 12 natural serotypes and 16 novel serotypes expressing multiple epitopes combinations that do not occur in nature. Importantly, a candidate GMMA displaying a hybrid OAg elicited broadly cross-bactericidal antibodies against a large panel of S. flexneri serotypes

Structural characterisation of novel exopolysaccharide biosynthesized by potential probiotic strain Lactobacillus fermentum D12

Lactobacillus fermentum D12, biosynthesize exopolysaccharides, is released in large amounts in MRS broth supplemented with glucose. High Performance Size Exclusion Chromatography (HPSEC), 1H-NMR, GC and GC-MS analysis revealed that this strain produces three different types of EPOLs; the first homopolysaccharide (HoPOL) of a molecular weight of 400 kDa and two different low molecular weight heteropolysaccharides (HePOLs) of less than 2 kDa. 2D-NMR spectroscopy analysis revealed that HoEPOL, with the highest molecular mass, is composed of repeating units of D-glucose linked by an α-1,4-glycosidic bond, where 20% of the glucose subunits is acetylated at C-3. Further chromatographic analyses and NMR experiments showed that each HePOL contained mannose, glucose and galactose in an averaged relative molar ratio of 1.78:0.87:1 and 6.38:1.6:1, respectively. Since a probiotic strain survival in rigorous gastrointestinal (GI) conditions is the first probiotic selection criterion to be met, and with respect to efficient survival of D12 strain in GIT in vitro (bacteria counts ≥106 CFU ml-1) the potential probiotic role of Lb. fermentum D12 was evaluated. Also, sensitivity to different antibiotics, minimum inhibitory concentrations (MICs), antagonistic activity and analysis of fermentation of different carbohydrates using API 50 CHL media of this potential probiotic strain was assessed

Genetic and structural elucidation of capsular polysaccharides from Streptococcus pneumoniae serotype 23A and 23B, and comparison to serotype 23F

Streptococcus pneumoniae is a globally important encapsulated human pathogen with approximately 100 different serotypes recognized. Serogroup 23 consists of serotype 23F, present in licensed vaccines, and emerging serotypes 23A and 23B. Here, we report the previously unknown structures of the pneumococcal capsular polysaccharides serotype 23A and 23B determined using genetic analysis, NMR spectroscopy, composition and linkage analysis and Smith degradation (of polysaccharide 23A). The structure of the serotype 23A capsular polysaccharide is: \u21924)-\u3b2-D-Glcp-(1\u21923)-[[\u3b1-L-Rhap-(1\u21922)]-[Gro-(2\u2192P\u21923)]-\u3b2-D-Galp-(1\u21924)]-\u3b2-L-Rhap-(1\u2192. This structure differs from polysaccharide 23F as it features a disaccharide backbone and the di-substituted \u3b2-Gal is linked to \u3b2-Rha as a side chain. This is due to the different polymerization position catalysed by the unusually divergent repeat unit polymerase Wzy in the 23A cps biosynthesis locus. Steric crowding in 23A, confirmed by molecular models, causes the NMR signal for H-1 of the di-substituted 2,3-\u3b2-Gal to resonate in the \u3b1-anomeric region. The structure of the serotype 23B capsular polysaccharide is the same as 23F, but without the terminal \u3b1-Rha: \u21924)-\u3b2-D-Glcp-(1\u21924)-[Gro-(2\u2192P\u21923)]-\u3b2-D-Galp-(1\u21924)-\u3b2-L-Rhap-(1\u2192. The immunodominant terminal \u3b1-Rha of 23F is more sterically crowded in 23A and absent in 23B. This may explain the reported typing cross reactions for serotype 23F: slight with 23A and none with 23B

GMMA and Glycoconjugate Approaches Compared in Mice for the Development of a Vaccine against <i>Shigella flexneri</i> Serotype 6

Shigella infections are one of the top causes of diarrhea throughout the world, with Shigella flexneri being predominant in developing countries. Currently, no vaccines are widely available and increasing levels of multidrug-resistance make Shigella a high priority for vaccine development. The serotype-specific O-antigen moiety of Shigella lipopolysaccharide has been recognized as a key target for protective immunity, and many O-antigen based candidate vaccines are in development. Recently, the Generalized Modules for Membrane Antigens (GMMA) technology has been proposed as an alternative approach to traditional glycoconjugate vaccines for O-antigen delivery. Here, these two technologies are compared for a vaccine against S. flexneri serotype 6. Genetic strategies for GMMA production, conjugation approaches for linkage of the O-antigen to CRM197 carrier protein, and a large panel of analytical methods for full vaccine characterization have been put in place. In a head-to-head immunogenicity study in mice, GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel. When formulated on Alhydrogel, GMMA and glycoconjugate elicited similar levels of persistent anti-O-antigen IgG with bactericidal activity. Glycoconjugates are a well-established bacterial vaccine approach, but can be costly, particularly when multicomponent preparations are required. With similar immunogenicity and a simpler manufacturing process, GMMA are a promising strategy for the development of a vaccine against Shigella

Structural characterisation of novel exopolysaccharide biosynthesized by potential probiotic strain Lactobacillus fermentum D12

Lactobacillus fermentum D12, biosynthesize exopolysaccharides, is released in large amounts in MRS broth supplemented with glucose. High Performance Size Exclusion Chromatography (HPSEC), 1H-NMR, GC and GC-MS analysis revealed that this strain produces three different types of EPOLs; the first homopolysaccharide (HoPOL) of a molecular weight of 400 kDa and two different low molecular weight heteropolysaccharides (HePOLs) of less than 2 kDa. 2D-NMR spectroscopy analysis revealed that HoEPOL, with the highest molecular mass, is composed of repeating units of D-glucose linked by an α-1,4-glycosidic bond, where 20% of the glucose subunits is acetylated at C-3. Further chromatographic analyses and NMR experiments showed that each HePOL contained mannose, glucose and galactose in an averaged relative molar ratio of 1.78:0.87:1 and 6.38:1.6:1, respectively. Since a probiotic strain survival in rigorous gastrointestinal (GI) conditions is the first probiotic selection criterion to be met, and with respect to efficient survival of D12 strain in GIT in vitro (bacteria counts ≥106 CFU ml-1) the potential probiotic role of Lb. fermentum D12 was evaluated. Also, sensitivity to different antibiotics, minimum inhibitory concentrations (MICs), antagonistic activity and analysis of fermentation of different carbohydrates using API 50 CHL media of this potential probiotic strain was assessed